To obtain clear images in microscopy it is critical to have an even field of illumination. However, as generally known, the field of illumination may be illuminated more brightly near the center of the specimen with the light intensity diminishing radially toward the perimeter of the illumination field. This change in lighting, known as vignetting, presents difficulty in the analysis of specimen images acquired in the field.
Unfortunately, the problem of vignetting affects almost every microscope. Vignetting is most often the result of a shadowing effect, caused by the finite width of optical elements and apertures within the illumination and imaging optical paths. It is also impacted by aberrations within each set of lenses. All of these sources of vignetting typically combine together to create a non-negligible background intensity variation across the image plane.
Attempts have been made to correct for vignetting by providing a diffuser after the illumination source and before the specimen. This diffuser may be ground glass, a Fresnel lens, or a fly's eye lens among others. Instead of altering the illumination source, an alternate method of correcting for vignetting includes applying an algorithm to an image generated with lighting suffering from vignetting to artificially correct the image to address vignetting. This method involves altering the image taken under illumination suffering from vignetting—it does not correct the vignetting itself. In both cases it is difficult to eliminate all effects of the lighting variation resulting in vignetting.
There is a need in the art for apparatus and methods to provide a more consistent intensity of illuminating light across the entire field of view of a microscope at the imaging plane.